Problem with paraffin block slicing - (Dec/01/2010 )
my lab has been having some issues with our histology protocol. In trying to optimize to save reagents and time, we have so far have been using the following
Step 1: Dehydration
1. Remove sample from formaldehyde and immerse in distilled H2O 30 min
2. Transfer sample to 70% ethanol 30 min
3. Transfer sample to 80% ethanol 30 min
4. Transfer sample to 95% ethanol 30 min
5. Transfer sample to 100% ethanol (Batch 1) 30 min
6. Transfer sample to 100% ethanol (Batch 2) 30 min
7. Transfer sample to Xylene Batch 1 30 min
8. Transfer sample to Xylene Batch 2 30 min
9. Transfer sample to paraffin boat I in the oven (preheated at 65°C) 40 min
10. Transfer sample to paraffin boat II 40 min
11. Transfer sample to paraffin boat III 40min
Step 2: Paraffin embedding
1. Paraffin dispenser should be preheated several hours in advance.
2. Hold the specimen with tweezers in desired position at the bottom of the metal tray and pour paraffin over the sample until it is embedded.
Now here is where we run into problems. When slicing the embedded sample in a microtome, the sample is lost from the paraffin slices (due to being too brittle I guess?) Does anyone have any idea how to modify the dehydration protocol to prevent this from happening? Maybe less time in xylene?
This has been an ongoing issue, so any advice would be greatly appreciated!
You have been waiting about 2 weeks for help. Maybe a short phone call to local hospital histology lab to ask your question??
try here: http://www.ihcworld.com/_faq/histology-faq/histology-faq.htm
I am new to Histology as well. But as far as I know, the Ethanol higher than 70% and Xylene are the steps to make your sample brittle. So I suppose that you can optimize the during of these steps. Try to keep Xylene steps long enough to allow Paraffin penetrate in but short enough to prevent sample hardening.
SOOO many possibilities..
it depends a lot on the tissue you are using, the size of the tissue and making sure there is enough wax around it, the type of wax (parrafin), your skill with the microtome and the thickness you are cutting at.
if youve never done histology before you REALLY need a pro to show you the ropes - it isnt something you can learn from a book.
id recomend getting in touch with a pathology lab and asking if you can watch for an hour
how you make parraffin block seems to be fine with me.
I think that the problem is either your tissue (fixation, size) or how you cut the section.